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1.
Primitive blood cells differentiate from the ventral mesoderm blood islands in Xenopus embryos. In order to determine the tissue interactions that propagate blood formation in early embryogenesis, we used embryos that had the ventral cytoplasm removed. These embryos gastrulated normally, formed a mesodermal layer and lacked axial structures, but displayed a marked enhancement of alpha-globin expression. Early ventral markers, such as msx-1, vent-1 and vent-2 were highly expressed at the gastrula stage, while a dorsal marker, goosecoid, was diminished. Several lines of experimental evidence demonstrate the critical role of animal pole-derived ectoderm in blood cell formation: 1) Mesoderm derived from dorsal blastomeres injected with beta-galactosidase mRNA (as a lineage tracer) expressed alpha-globin when interfaced with an animal pole-derived ectodermal layer; 2) Embryos in which the animal pole tissue had been removed by dissection at the blastula stage failed to express alpha-globin; 3) Exogastrulated embryos that lacked an interaction between the mesodermal and ectodermal layers failed to form blood cells, while muscle cells were observed in these embryos. Using dominant-negative forms of the BMP-4 and ALK-4 receptors, we showed that activin and BMP-4 signaling is necessary for blood cell differentiation in ventral marginal zone explants, while FGF signaling is not essential. In ventralized embryos, inactivation of the BMP-4 signal within a localized area of the ectoderm led to suppression of globin expression in the adjacent mesoderm layer, but inactivation of the activin signal did not have this effect. These observations suggest that mesodermal cells, derived from a default pathway that is induced by the activin signal, need an additional BMP-4-dependent factor from the overlying ectoderm for further differentiation into a blood cell lineage.  相似文献   

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In early vertebrate development, mesoderm induction is a crucial event regulated by several factors including the activin, BMP and FGF signaling pathways. While the requirement of FGF in Nodal/activin-induced mesoderm formation has been reported, the fate of the tissue modulated by these signals is not fully understood. Here, we examined the fate of tissues when exogenous activin was added and FGF signaling was inhibited in animal cap explants of Xenopus embryos. Activin-induced dorsal mesoderm was converted to ventral mesoderm by inhibition of FGF signaling. We also found that inhibiting FGF signaling in the dorsal marginal zone, in vegetal-animal cap conjugates or in the presence of the activin signaling component Smad2, converted dorsal mesoderm to ventral mesoderm. The expression and promoter activities of a BMP responsive molecule, PV.1 and a Spemann organizer, noggin, were investigated while FGF signaling was inhibited. PV.1 expression increased, while noggin decreased. In addition, inhibiting BMP-4 signaling abolished ventral mesoderm formation induced by exogenous activin and FGF inhibition. Taken together, these results suggest that the formation of dorso-ventral mesoderm in early Xenopus embryos is regulated by a combination of FGF, activin and BMP signaling.  相似文献   

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Summary Two nuclear markers were used to investigate the origin of cells in secondary embryos ofXenopus induced by dorsal lip transplants, and to determine the ability of the chordomesoderm to direct cells to change their fates.3H-thymidine was used to label cells transplanted between individualX. laevis embryos, and nuclear quinacrine fluorescence was used to distinguishX. borealis tissues transplanted toX. laevis hosts. In the first set of experiments, dorsal lip tissue (also known as the dorsal marginal zone; DMZ) was transplanted to the ventral marginal zone (VMZ) of host embryos. The marginal zone is the toroid of presumptive mesodermal cells which involutes during gastrulation. Examination of the secondary embryos resulting from these grafts revealed that their notochords were derived almost exclusively from transplanted cells whereas their nervous systems and somites were composed almost entirely of host cells. Next, the nuclear markers were used to show the normal fates of the tissue of the ventral equatorial region immediately above the VMZ by orthotopic grafting. This tissue was found to give rise to structures in the ventral posterior portions of the tailbud embryo. Finally, the same ventral tissue was labeled and transplanted to the dorsal equatorial region above the DMZ. As a result, it was induced to change its fate and become neural. These results lend unequivocal support to Spemann's theory of neural induction which has recently been questioned.  相似文献   

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Ventral blood island mesoderm and dorsal lateral plate mesoderm were removed from Rana pipiens embryos at successive developmental stages (stages 13-19; 50-118 h) and cultured as individual explants in serum-free medium. After 5-7 days, the cultures were harvested, and differential counts were made of Wright-Giemsa-stained cells. Ventral blood island explants gave rise to cells of the myeloid lineage, suggesting that ventral blood island mesoderm was committed to hemopoiesis at the time of explant. Although erythrocytes were present in the cultures, granulocytes and monocyte/macrophages predominated. This differentiation profile occurred without the addition of any exogenous humoral factors. Monocyte/macrophages and immature precursor cells exhibited recurring inverse fluctuations with respect to one another. In all cases examined, cultures of dorsal lateral plate mesoderm showed marginal hemopoietic cell differentiation, suggesting a requirement for exogenous humoral factors and/or cell-cell interactions. When viewed in the context of previous studies from our laboratory, these results demonstrate that, in the amphibian embryo, there are two sources of hemopoietic stem cells separated both in space and time.  相似文献   

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We have investigated the properties of the epithelial layer of the dorsal marginal zone (DMZ) of the Xenopus laevis early gastrula and found that it has inductive properties similar to those of the entire Spemann organizer. When grafts of the epithelial layer of the DMZ of early gastrulae labelled with fluorescein dextran were transplanted to the ventral sides of unlabelled host embryos, they induced secondary axes composed of notochord, somites and posterior neural tube. The organizer epithelium rescued embryos ventralized by UV irradiation, inducing notochord, somites and posterior neural tube in these embryos, while over 90% of ventralized controls showed no such structures. Combinations of organizer epithelium and ventral marginal zone (VMZ) in explants of the early gastrula resulted in convergence, extension and differentiation of dorsal mesodermal tissues, whereas similar recombinants of nonorganizer epithelium and the VMZ did none of these things. In all cases, the axial structures forming in response to epithelial grafts were composed of labelled graft and unlabelled host cells, indicating an induction by the organizer epithelium of dorsal, axial morphogenesis and tissue differentiation among mesodermal cells that otherwise showed non-axial development. Serial sectioning and scanning electron microscopy of control grafts shows that the epithelial organizer effect occurs in the absence of contaminating deep cells adhering to the epithelial grafts. However, labelled organizer epithelium grafted to the superficial cell layer contributed cells to deep mesodermal tissues, and organizer epithelium developed into mesodermal tissues when deliberately grafted into the deep region. This shows that these prospective endodermal epithelial cells are able to contribute to mesodermal, mesenchymal tissues when they move or are moved into the deep environment. These results suggest that in normal development, the endodermal epithelium may influence some aspects of the cell motility underlying the mediolateral intercalation (see Shih, J. and Keller, R. (1992) Development 116, 901-914), as well as the tissue differentiation of mesodermal cells. These results have implications for the analysis of mesoderm induction and for analysis of variations in the differentiation and morphogenetic function of the marginal zone in different species of amphibians.  相似文献   

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We have studied the response of Xenopus blastula ectoderm to fibroblast growth factor and to lithium ion. The properties of acidic and basic FGF are very similar showing a 50% induction level at 1-2 ng ml-1 and a progressive increase of muscle formation up to concentrations of 100-200 ng ml-1. The elongation of explants also shows a dose-response relationship. The minimum contact requirement for induction of ectoderm explants is about 90 min and the stage range of ectodermal competence extends from midblastula to early gastrula, both these figures resembling those obtained in embryological experiments with vegetal tissue as the inducer. Lithium chloride concentrations which produce anteriorization of whole embryos have no effect on isolated ectoderms unless accompanied by FGF. Simultaneous treatment with FGF and Li lead to a marked enhancement of both elongation and muscle formation over that produced by FGF alone. By contrast, ventral marginal explants show increased elongation and muscle formation if treated with lithium alone suggesting that they have already received a low-dose FGF treatment within the embryo. It is concluded that endogenous FGF may be solely responsible for inducing the ventral mesoderm and that dorsalization of ventral mesoderm to the level of somitic muscle might be achieved either by a very high local concentration of FGF in the dorsal region, or by the action of a second, synergistic, agent in the dorsal region.  相似文献   

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Horseradish peroxidase (HRP) was used as an intracellular lineage tracer in two experiments designed to reveal the sites of origin of cells that formed the duplicate embryo which developed in relation to an organizer grafted in the ventral marginal zone (VMZ) of Xenopus laevis embryos. In the first experiment a dorsal blastoporal lip fully labeled with HRP was grafted in the VMZ of an unlabeled embryo at the beginning of gastrulation. This resulted in development of a second embryo in which labeled cells, of graft origin, formed the notochord, and parts of the somites, endoderm, and neural tube. The second experiment was designed to show the sites of origin of the host's cells that formed parts of the induced embryo. HRP was injected into individual blastomeres in a series of Xenopus embryos at the 32-cell stage and each embryo received an unlabeled organizer graft in the VMZ at the beginning of gastrulation. In these embryos the lineages that contributed to the host's primary neural tube did not contribute any cells to the induced neural tube. All the cells in the induced neural tube which originated from the host were descendants of ventral blastomeres that did not contribute to the neural tube normally. This shows that the second neural tube is formed as a result of the action of the organizer on cells in its immediate vicinity which would not normally have entered neural pathways of differentiation.  相似文献   

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Studies of morphogenesis in early Xenopus embryos have focused primarily on gastrulation and neurulation. Immediately following these stages is another period of intense morphogenetic activity, the neurula-to-tailbud transition. During this period the embryo is transformed from the spherical shape of the early stages into the long, thin shape of the tailbud stages. While gastrulation and neurulation depend largely on active cell rearrangement and cell shape changes in dorsal tissues, we find that the neurula-to-tailbud transition depends in part on activities of ventral cells. Ventral explants of neurula lengthen autonomously as much as the ventral sides of intact embryos, while dorsal explants lengthen less than the dorsal sides of intact embryos. Analyses of cell division, cell shapes, and cell rearrangement by transplantation of labeled cells and by time lapse recordings in live intact embryos concur that cell rearrangements in ventral mesoderm and ectoderm contribute to the autonomous anterior-posterior axis lengthening of ventral explants between neurula and tailbud stages.  相似文献   

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The interplay of fibroblast growth factor (FGF) and nodal signaling in the Xenopus gastrula marginal zone specifies distinct populations of presumptive mesodermal cells. Cells in the vegetal marginal zone, making up the presumptive leading edge mesoderm, are exposed to nodal signaling, as evidenced by SMAD2 activation, but do not appear to be exposed to FGF signaling, as evidenced by the lack of MAP kinase (MAPK) activation. However, in the animal marginal zone, activation of both SMAD2 and MAPK occurs. The differential activation of these two signaling pathways in the marginal zone results in the vegetal and animal marginal zones expressing different genes at gastrulation, and subsequently having different fates, with the vegetal marginal zone contributing to ventral mesoderm (e.g. ventral blood island) and the animal marginal zone giving rise to dorsal fates (e.g. notochord and somite). We report here the cloning of a cDNA encoding a novel nuclear protein, Xmenf, that is expressed in the vegetal marginal zone. The expression of Xmenf is induced by nodal signaling and negatively regulated by FGF signaling. Results from animal cap studies indicate that Xmenf plays a role in the pathway of ventral mesoderm induction in the vegetal marginal zone.  相似文献   

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To elucidate the mechanism of determination and regulation of hemopoiesis in the early Xenopus embryo, explants of dorsal and ventral mesoderm from various stage embryos were cultured alone or combined with various tissues derived from the same stage embryo. Western blot analysis of larvae-specific globin expression using monoclonal antibody L5.41 revealed that extensive erythropoiesis occurred in the explants of ventral mesoderm from st. 22 tailbud embryo, but not in those of dorsal mesoderm. Experiments using combined explants at this stage demonstrated that the in vitro differentiation of erythrocytes in the ventral mesoderm could be completely inhibited by the dorsal tissue, including neural tube, notochord, and somite mesoderm, but not by other mesoderms, gut endoderm, or forebrain. Subsequent explant studies showed that the notochord alone is sufficient for this inhibition. Furthermore, the ventral mesoderm explant from the st. 10+ early gastrula embryo was not able to differentiate into erythroid cells. However, small amounts of globin were expressed if ventral mesoderm of this stage was combined with animal pole cells which were mainly differentiated to epidermis. This stimulation was enhanced when both tissues were excised together without separation, while none of the other parts of st. 10+ embryo had this stimulatory effect. These observations found in the combined explants suggest that in vivo interactions between the ventral mesoderm and adjacent tissues are important for normal development of erythroid precursor cells.  相似文献   

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Fibroblast growth factor (FGF) is established as an initiator of signaling events critical for neurogenesis and mesoderm formation during early Xenopus embryogenesis. However, less is known about the role FGF signaling plays in endoderm specification. Here, we show for the first time that endoderm-specific genes are induced when FGF signaling is blocked in animal cap explants. This block of FGF signaling is also responsible for a significant enhancement of endodermal gene expression in animal cap explants that are injected with a dominant-negative BMP-4 receptor (DNBR) RNA or treated with activin, however, neural and mesoderm gene expression is diminished. Consistent with these results, the injection of dominant-negative FGF receptor (DNFR) RNA expands endodermal cell fate boundaries while FGF treatment dramatically reduces endoderm in whole embryos. Taken together, these results indicate that inhibition of FGF signaling promotes endoderm formation, whereas the presence of active FGF signaling is necessary for neurogenesis/mesoderm formation.  相似文献   

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When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.  相似文献   

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Gastrulation in vertebrates is a highly dynamic process driven by convergent extension movements of internal mesodermal cells, under the regulatory activity of the Spemann-Mangold or gastrula organizer. In a large-scale screen for genes expressed in the organizer, we have isolated a novel gene, termed Xmc, an acronym for Xenopus marginal coil. Xmc encodes a protein containing two widely spaced evolutionarily non-conserved coiled coils. Xmc protein is found in vesicular aggregates in the cytoplasm and associated with the inner plasma membrane. We show that Xmc is expressed in a dynamic fashion around the blastoporal circumference, in mesodermal cells undergoing morphogenetic movements, in a pattern similar to FGF target genes. Likewise, Xmc expression can be induced by ectopic XeFGF signaling and the early mesodermal expression is dependent on FGF receptor-mediated signaling. Morpholino-mediated translational 'knock-down' of Xmc results in embryos that display a reduced elongation of the antero-posterior axis and in a pronounced inhibition of morphogenetic movements in embryos and dorsal marginal zone explants. Xmc loss-of-function does not interfere with mesoderm induction or maintenance per se. Our results suggest that Xmc is a novel FGF target gene that is required for morphogenetic movements during gastrulation in Xenopus.  相似文献   

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